The present invention relates to a storage-stable liquid formulation comprising a laccase, a method for improving the storage-stability of liquid formulations comprising a laccase and the use of said liquid formulations for a personal care application or for bleaching or for textile applications such as dyeing of fabrics.
Industrial enzymes have generally been formulated as particulate solids (e.g. in powder or granulated form, optionally with a coating of some kind) or in the form of a water-based solution.
A number of solid formulations (e.g. enzyme powders) have the disadvantages that dust formation readily takes place, whichxe2x80x94unless special precautions are taken may result in contamination of the surrounding environment and thereby pose a risk to the health of persons handling such compositions.
Water-based, liquid enzyme formulations essentially eliminate the risk of dust formation. Owing to the fact that practically all enzymes except exert their activity in the presence of water it is generally not feasible to prepare storage-stable formulations comprising free (e.g. unencapsulated or uncoated) enzymes.
Currently available water-based liquid enzyme formulations have a relatively short time span, within which it is possible to operate, as the residual enzymatic activity is reduced to an unacceptable low level after a few weeks of storage if not stored under cooling.
Feng Xu et al, (1996), Biochimica et Biophysica Acta, 1292, p. 303-311 shows that the stability of various laccases is dependent on the pH and the temperature after pre-incubation with ABTS for 1 hour.
Palmiert et al., (1993), Applied Microbiol. Biotechnol., 39, p. 632-636, shows that a specific laccase incubated at 25xc2x0 C. is more stable at pH 7 than at pH 3 for a period of 300 minutes (i.e. 6 hours).
Thus, there is a need for liquid formulations comprising a laccase which are storage-stable for a period of more than one day. The present invention provides formulations fulfilling such a need.
It is the object of the present invention to provide a storage-stable liquid formulation comprising a laccase which is storage-stable at 10-60xc2x0 C. for a prolonged period of time.
A liquid formulation comprising (i) a laccase and (ii) at least one polyalcohol, which formulation has a pH which is more alkaline than the pH optimum of the laccase, has an improved storage stability or shelf-life in comparison to corresponding formulation without (a) polyalcohol(s) with a pH at the pH optimum of the laccase in question.
Laccases (benzenediol:oxygen oxidoreductases) (E.C. class 1.10.3.2 according to Enzyme Nomenclature (1992) Academic Press, Inc.) are multi-copper containing enzymes that catalyse the oxidation of phenols. Laccase-mediated oxidation results in the production of aryloxy-radical intermediates from suitable phenolic substrates; the ultimate coupling of the intermediates so produced provides a combination of dimeric, oligomeric, and polymeric reaction products. Certain reaction products may be used to form dyes suitable for dyeing keratinous fibres, such as hair, wool and textiles.
Laccases are obtainable from a variety of microbial sources, notably bacteria and fungi (including filamentous fungi and yeasts), and suitable examples of laccases are to found among those obtainable from fungi, including laccases obtainable from strains of Aspergillus, Neurospora (e.g. N. crassa), Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Trametes [some species/strains of which are known by various names and/or have previously been classified within other genera; e.g. Trametes villosa=T. pinsitus=Polyporus pinsitis (also known as P. pinsuitus or P. villosus)=Coriolus pinsitus], Polyporus, Rhizoctonia (e.g. R. solani), Coprinus (e.g. C. plicatilis, Coprinus cinereus), Psatyrella, Myceliophthora (e.g. M. thermophila), Schytalidium, Phlebia (e.g. P. radita; see WO 92/01046), Coriolus (e.g. C.hirsutus; see JP 2-238885), Pyricularia or Rigidoporus.
Preferred laccases in the context of the invention include laccases obtainable from Myceliophthora thermophila and laccase obtainable from Polyporus pinsitus. 
A liquid formulation of the invention may or may not contain other ingredients. Other ingredients contemplated include laccase substrate or mediator, pH regulating agents, anti-microbial agents, dispersing agents and/or viscosity-regulating agents.
The invention also relates to a method for improving the storage-stability of a laccase by storing the laccase in the presence of a polyalcohol at a pH,which is 0.5 to 5.5 pH units more alkaline than the pH optimum of the laccase.
Finally the invention relates to the use of a liquid formulation of the invention for personal care applications, such as for hair dying, bleaching and for textile application such as dyeing of fabrics.